Growth promoting polypeptides and preparation method

Abstract

The application discloses (A) a growth-promoting polypeptide having molecular weight from about 5,000 to about 7,000 chromatographic mobility under electrophoresis from about 0.25 to about 0.37 relative to lysine at pH 5 and from about 0.17 to about 0.43 relative to aspartic acid at pH 7.5, terminal amino acid at its amino end being asparagine or aspartic acid, and respective amounts of aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, leucine, tyrosine, phenylalanine, lysine, arginine, trypophan, and cysteine or carboxymethyl cysteine, and also a couple of instances also methionine, isoleucine and histidine; and (B) the derivation of the polypeptides from blood serum or plasma or a plasma fraction by combined steps of (a) homogenizing the starting material in water, (b) admixing a solution of hydrochloric acid in ethanol thereby splitting the polypeptide from its carrier proteins and precipitating them, (c) adjusting the pH to 8.4 to provide a precipitate, (d) separating it, (e) adjusting the pH of the liquid residue to 3, (f) admixing a water-insoluble strongly acidic cation exchange matrix of sulfopropyl-oxygen-linked-substitued dextran cross-linked with epichlorhydrin at pH 3, (g) separating the gel matrix from the liquid, (h) eluting the polypeptide-containing material from the matrix with (x) ammonium acetate at pH 10 or (y) 0.75M ammonium bicarbonate; and (i) precipitating the polypeptide by admixing acetone.

What is claimed is:  pg,18 
     
       1. A growth-promoting polypeptide derivable from mammalian blood plasma by a procedure which includes subjecting the plasma material to an acid, bond-splitting agent; and having (i) molecular weight in the range of from about 5,000 to about 7,000 determined by gel filtration enhanced by using insulin of its known molecular weight as a standard for comparison, (ii) chromatographic mobility under electrophoresis relative to lysine at pH 5 of from about 0.25 to about 0.37 and relative to aspartic acid at pH 7.5 of from about 0.17 to about 0.43, and (iii) the terminal amino acid at its amino end is asparagine or aspartic acid, and also having the respective constitution provided by one of the following groups (a), (b), and (c) of combined amino acids, with each said acid being in its respectively noted molar content: a. aspartic acid 4.1, threonine 4.3, serine 7.8, glutamic acid 5.1, proline 8.2, glycine 10.2, alanine 4.9, valine 4.0, methionine 0.3, isoleucine 1.3, leucine 2.1, tyrosine 0.6, phenylalanine 1.1, histidine 1.5, lysine 2.8, arginine 4.0, tryptophan 1.0, and carboxymethyl cysteine 1.0, per mol of said polypeptide;   b. aspartic acid 4.8, threonine 1.9, serine 3.5, glutamic acid 4.6, proline 7.3, glycine 7.1, alanine 7.0, valine 4.9, methionine 0.9, isoleucine 0.8, leucine 3.3, tyrosine 0.9, phenylalanine 2.2, histidine 3.8, lysine 2.5, arginine 4.6, tryptophan 1.0, and cysteine (determined as cysteic acid) 1.14, per mol of said polypeptide; and   c. aspartic acid 5.2, threonine 4.0, serine 3.1, glutamic acid 8.1, proline 0.8, glycine 2.1, alanine 1.1, valine 1.8, leucine 1.0, tryosine 2.9, phenylalanine 1.0, lysine 3.8, arginine 1.3, tryptophan 1.0, and carboxymethyl cysteine 8.0, per mol of said polypeptide.   
     
     
       2. The growth-promoting polypeptide as claimed in claim 1, having (i) molecular weight of about 7,000, (ii) mobility under elctrophoresis relative to lysine at pH 5 of 0.29 and relative to aspartic acid at pH 7.5 of 0.17, (iii) asparagine as its said amino end terminal amino acid; and (iv) the composition provided by the following combined amino acids each in its respectively noted molar content: aspartic acid 4.1, threonine 4.3, serine 7.8, glutamic acid 5.1, proline 8.2, glycine 10.2, alanine 4.9, valine 4.0, methionine 0.3, isoleucine 1.3, leucine 2.1, tyrosine 0.6, phenylalanine 1.1, histidine 1.5, lysine 2.8, arginine 4.0, tryptophan 1.0, and carboxymethyl cysteine 1.0. 
     
     
       3. The growth-promoting polypeptide as claimed in claim 1 having (i) molecular weight of about 7,000, (ii) mobility under electrophoresis relative to lysine at 5 of 0.37, (ii) asparagine as its said amino end terminal amino acid; and (iv) the composition provided by the following combined amino acids each in its respectively noted molar content: aspartic acid 4.8, threonine 1.9, serine 3.5, glutamic acid 4.6, proline 7.3, glycine 7.1, alanine 7.0, valine 4.9, methionine 0.9, isoleucine 0.8, leucine 3.3, tyrosine 0.9, phenylalanine 2.2, histidine 3.8, lysine 2.5, arginine 4.6, tryptophan 1.0, and cysteine (determined as cysteic acid) 1.14, per mol of said polypeptide. 
     
     
       4. The growth-promoting polypeptide as claimed in claim 1, having (i) molecular weight of about 5,000, (ii) mobility under electrophoresis relative to aspartic acid at pH 5 of 0.25 and at pH 7.5 of 0.43, (iii) aspartic acid as its said amino end terminal amino acid; and (iv) the composition provided by the following combined amino acids each in its respectively noted molar content: aspartic acid 5.2, threonine 4.0, serine 3.1, glutamic acid 8.1, proline 0.8, glycine 2.1, alanine 1.1, valine 1.8, leucine 1.0, tryosine 2.9, phenylalanine 1.0, lysine 3.8, arginine 1.3, tryptophan 1.0, and carboxymethyl cysteine 8.0. per mol of said polypeptide. 
     
     
       5. In the method of deriving a growth-promoting polypeptide having molecular weight in the range of from about 5,000 to about 7,000, chromatographic mobility under electrophoresis relative to lysine at pH 5 of from about 0.25 to about 0.37 and relative to aspartic acid at pH 7.5 of from about 0.17 to about 0.43, and the terminal amino acid at its amino end being asparagine or aspartic acid, from mammalian blood serum or plasma or a plasma fraction from which said polypeptide can be derived, the combination of steps comprising (a) homogenizing the Cohn et al. plasma fraction IV or fraction IV-6+7 with sufficient water and for a time sufficient to homogenize said fraction, (b) subjecting the resulting homogenate with a sufficient polypeptide-to-protein-linkage -bond-splitting amount of a mixture of about 1 part of concentrated hydrochloric acid in about forty parts of about 96% ethanol at about 0° C for a time sufficient to split said polypeptide from its carrier proteins and to precipitate said proteins, (c) adjusting the pH of the reaction mixture to about 8.4 with the required amount of a compatible water-soluble alkali metal hydroxide, (d) separating the precipitate from the liquid vehicle, (e) adjusting the pH of said liquid vehicle to about 3 by cautiously admixing it with concentrated hydrochloric acid, (f) admixing the resulting acid liquid vehicle at about 0° C with a polypeptide-adsorbing sufficient amount of a finely divided, water-insoluble strongly acidic cation exchange gel matrix constituted of a three-dimensional network of sulfopropyl-oxygen-linked-substituted dextran chains cross-linked with epichlorhydrin equilibrated with sodium chloride at about pH 3, for a time sufficient to adsorb said polypeptide; (g) separating said water-soluble gel matrix with said adsorbed polypeptide from its surrounding liquid vehicle, (h) eluting the polypeptide containing material from said gel matrix with (x) about 0.2M ammonium acetate adjusted to pH 10 by addition of about 25% ammonium hydroxide, or (y) about 0.75M ammonium bicarbonate; and (i) precipitating the crude polypeptide from the resulting eluate by admixing the latter at about -15° C with about 4 times its volume of acetone. 
     
     
       6. The combination of steps as claimed in claim 5, wherein said crude polypeptide precipitate is separated from said alkaline acetone solution. 
     
     
       7. The combination of steps as claimed in claim 6, wherein said separated crude polypeptide precipitate is extracted with a growth promotion polypeptides extraction effective amount of an about 20% formic acid aqueous solution to extract the growth promotion polypeptides from said precipitate; the resulting extract of said polypeptides in said formic acid solution then is separated from the aqueous acetone-insoluble residue of said precipitate and subjected to gelfiltration at about 3° C through a gelfiltration column packed with a finely divided water-insoluble gel matrix constituted of a three-dimensional network of dextran chains cross-linked with epichlorhydrin. 
     
     
       8. The combination of steps as claimed in claim 7, wherein said finely divided gel matrix has a particle size of from about 50 to 150 microns and a water regain of 5±0.3 ml. of water per gm. of dry particles, and effluent fractions of 25 to 30 ml. each are collected at a flow rate of about 300 ml. per hour. 
     
     
       9. The combination of steps as claimed in claim 7, wherein said finely divided gel matrix has a particle size from about 40 to 120 and a water regain of from 7.5±0.5 ml. of water per gm. of dry particles; and said extract of said polypeptides is applied to said column at a flow rate of 4 ml. per sq. cm. per hour. 
     
     
       10. The combination of steps as claimed in claim 9, wherein the effluent from the gelfiltration therein is subjected to further gelfiltration through a column packed with a finely divided water-insoluble gel matrix constituted of a three-dimensional network of dextran chains cross-linked with epichlorhydrin and having a particle size of from about 20 to about 80 microns and a water regain of 5±0.3 ml. of water per gm. of dry particles; and effluent fractions of 6 ml. each are collected from said column at a flow rate of 25 ml. per hour. 
     
     
       11. The combination of steps as claimed in claim 8, wherein from about 200 to about 300 mg. of the solute of the pooled fractions 150 to 200 of claim 8 are dissolved in 2 ml. of 0.025M N-ethylmorpholine acetate buffer and subjected to column zone electrophoresis in a column packed with zone electrophoresis grade cellulose powder, under about 800 volts and about 20 milliamperes and using 0.05M N-ethylmorpholine acetate buffer at about pH 7.5 as the elution solvent. 
     
     
       12. The combination of steps as claimed in claim 11, wherein serial fractions of 4 ml. each of the effluent from said electrophoresis are collected at a flow rate of 40 ml. per hour. 
     
     
       13. The combination of steps as claimed in claim 12, wherein from about 50 to about 200 mg. of the solute of either of pooled effluent fractions 45 to 55 or 57 to 66 of claim 12 are dissolved in per 2 ml. of 0.025M of pyridine acetate buffer and subjected to column zone electrophoresis by being applied to an analytical column packed with medium fiber length zone electrophoresis grade of cellulose powder, under about 1000 volts at about 10 milliamperes and using 0.05M pyridine acetate buffer at pH 5 as the elution solvent. 
     
     
       14. The combination of steps as claimed in claim 13, wherein serial fractions of 1 ml. each of column effluent are collected at a flow rate of 15 ml. per hour. 
     
     
       15. The combination of steps as claimed in claim 14, wherein from about 3 to about 25 mg. of the solute of either of the pooled fractions 15 to 18 or the pooled fractions 23 to 40 of claim 14 are dissolved in per one ml. of 0.02M hydrochloric acid and applied to an analytical gelfiltration column packed with the finely divided gel matrix constituted of a threedimensional network of dextran chains cross-linked with epichlorhydrin, in 0.02N hydrochloric acid, and subjected to gelfiltration at ambient temperature; and serial fractions of about 2.3 ml. are collected at a flow rate of 10 ml. per hour; and (i) effluent fractions 35 to 40 of said gelfiltration of the pooled effluent fractions 32 to 40 of claim 14 are pooled as one final product, and (ii) effluent fractions 35 to 40 of said gelfiltration of the pooled effluent fractions 23 to 31 of claim 14 are pooled as a second final product, and (iii) effluent fractions 38 to 44 of said gelfiltration of the pooled effluent fractions 15 to 18 of claim 14 are pooled as a third final product. 
     
     
       16. The combination of steps as claimed in claim 10, wherein from 200 to about 300 mg. of the solute of the pooled fractions 43 to 81 of claim 10 are dissolved in 2 ml. of 0.025M N-ethylmorpholine acetate buffer and subjected to column zone electrophoresis in a column packed with zone electrophoresis grade cellulose powder, under about 800 volts and about 20 milliamperes and using 0.05M N-ethylmorpholine acetate buffer at about pH 7.5 as the elution solvent.